Indian J Med Res 150, September 2019, pp 290-296 Quick Response Code:

DOI: 10.4103/ijmr.IJMR_1497_17

DNA damage induced by exposure to pesticides in children of rural

areas in Paraguay

Stela Benitez Leite1, Deidamia Mercedes Franco de Diana2, Jaime Alfredo Segovia Abreu2,
Domingo Santiago Avalos3, Marta Almada Denis4, Cristina Coronel Ovelar4, María José Samaniego Royg2,
Boris Alexei Thielmann Arbo2 & Ramón Corvalan4
Downloaded from http://journals.lww.com/ijmr by BhDMf5ePHKbH4TTImqenVA+lpWIIBvonhQl60Etgtdnn9T1vLQWJq3kbRMjK/ocE on 04/28/2022

1
Department of Community Medicine, Faculty of Health Sciences of Catholic University ‘Nuestra Señora de la
Asunción’ ,2Toxicological Genetics Laboratory of Faculty of Health Sciences of Catholic University ‘Nuestra
Señora de la Asunción’, 3Educational and Research Area of the General Directorate of Primary Health Care,
Ministry of Public Health & Social Welfare & 4Servicio Paz y Justicia Paraguay, Asuncion, Paraguay

Received September 19, 2017

Background & objectives: Chronic exposure to pesticides can damage DNA and lead to cancer, diabetes,
respiratory diseases and neurodegenerative and neurodevelopment disorders. The objective of this study
was to determine the frequency of DNA damage through the comet assay and micronucleus (MN) test in
two groups of children, under 10 yr of age living in rural Paraguay and in relation to pesticide exposure.
Methods: Two groups of 5 to 10 yr old children were formed; the exposed group (group A, n=43), born
and currently living in a community dedicated to family agriculture and surrounded by transgenic
soybean crops, and the control group (group B, n=41), born and living in a community dedicated to
family agriculture with biological control of pests. For each child, 2000 cells were studied for the MN test
and 200 cells for the comet assay.
Results: The comparison between exposed and control children revealed significant differences in
biomarkers studied for the measurement of genetic damage (cell death and DNA damage). The median
of MN was higher in the exposed group (6 vs. 1) (P<0.001). Binucleated cells (2.9 vs. 0.5, P<0.001); broken
eggs (5.5 vs. 1.0, P<0.001); karyorrhexis (6.7 vs. 0.5, P<0.001); kariolysis (14.0 vs. 1.0, P<0.001); pyknosis
(7.4 vs. 1.2, P<0.001) and condensed chromatin (25.5 vs. 7.0, P<0.001) were significantly higher in the
exposed group. The values of tail length (59.1 vs 37.2 µm); tail moment (TM) (32.8 vs. 14.4 µm); TM
olive (15.5 vs. 6); % DNA tail (45.2 vs. 27.6) and % DNA head (54.8 vs. 72.4), were significantly different
between the two groups.
Interpretations & conclusions: In children exposed to pesticides, a greater genotoxic and cytotoxic effect
was observed compared to non-exposed children. Our findings suggest that monitoring of genetic toxicity
in population exposed to pesticides and agrochemicals should be done.

Key words Biomonitoring - buccal micronucleus cytome assay - children - comet assay - DNA - pesticides

© 2019 Indian Journal of Medical Research, published by Wolters Kluwer - Medknow for Director-General, Indian Council of Medical Research
290
 LEITE et al: DNA DAMAGE IN CHILDREN 291
Paraguay is currently the world’s fourth largest
exporter and soy producer1. The area covered by this
crop covers 80 per cent of agricultural land. Between
August 2015 and July 2016, Paraguay officially
imported almost 38,000 tons of pesticides; 64 per
cent of the substances were herbicides, preferably
for the monoculture of transgenics2. The application
of agrochemicals is carried out in a context of low
and ineffective environmental regulation3. Pesticides
produce adverse effects on the environment and
health. There is evidence of the relationship between
exposure to pesticides and increased risk of cancers,
neurodegenerative disorders and neurodevelopment
and respiratory diseases and diabetes4. The importance
of early detection of genetic damage is that it allows
taking the necessary measures to reduce or suppress
the exposure to the deleterious agent when it is still
reversible and thereby prevent and reduce the risk
of developing neoplasia and other pathological
alterations5.
Micronucleus (MN) is a biomarker that is widely
used in biomonitoring studies to determine the
genetic risk associated with pesticide exposure. The
oral MN assay is a cytogenetic method to measure
genetic damage, cell proliferation, differentiation and
death in exfoliated oral cells6,7. The comet assay is a
sensitive technique to evaluate DNA damage of a
variety of damaged cells caused by different physical
and chemical agents. It is widely used in human
biomonitoring to measure DNA damage as a marker
of exposure to genotoxic agents or to investigate
genoprotective effects8.
Among the exposed populations, children
constitute a group that has particular exposure
characteristics and special vulnerability to
environmental toxics. Children may also differ from
adults in levels of detoxification, in processes of
DNA repair and cell proliferation. The most obvious
difference between children and adults is the reduced
impact of traditional confounding factors such as
cigarette smoking and occupational exposure5,9.
The San Juan colony, located in the district
of Francisco Alvarez Caballero, Department of
Canindeyú, Paraguay, was officially authorized by
the Rural Welfare Institute in 199710. At present,
there are about 450 families dedicated to family
agriculture. Surrounded by large areas of soybean,
mostly transgenic, the population is exposed to aerial
and terrestrial spraying, without any protection or
precaution towards people3. The objective of this study
was, therefore, to determine the frequency of DNA
damage (evaluated through the comet assay and MN
test in exfoliated cells of the buccal mucosa) and the
alteration of plasma cholinesterase and its relation to
pesticide exposures in two groups of children under
10 yr of age living in rural areas.
Material & Methods
This study was carried out at the Toxicological
Genetics Laboratory of Faculty of Health Sciences of
Catholic University “Nuestra Señora de la Asunción”
between January 2016 to June 2017, after the protocol
approval by the Ethics Committee of the Catholic
University, Asuncion, Paraguay. Two groups of healthy
children of 5-10 yr of age, of both sexes were formed.
Group A (n=43) was considered as exposed and group
B (n=41) was considered as not exposed. The exposed
population (group A) of healthy children comprised
those who were born in the place, and have been
living on a permanent basis on that location for more
than five years, of both sexes from 5 to 10 yr of age.
They were potentially exposed (chronic exposure) to
excessive and indiscriminate use11 of agrochemicals
used in intensive fumigation practices of monocultures
living less than 1 km away from the fumigated fields of
the San Juan Colony in the Department of Canindeyú
(Paraguay). This community was isolated, exposed
to the practices of aerial and terrestrial fumigation of
pesticides, and was surrounded by transgenic soybean
crops. Fumigation with pesticides took place every
15 days during 150 days when the soybean crops
mature (September-January); in the rainy season, the
fumigation cycle for the transgenic soybeans was
duplicated and further proceeded during the year due
to the existence of transgenic corn and transgenic
sunflower. The community had 450 families or less,
mostly living on family agriculture for self consumption
and as a means of income.
Unexposed population (group B) included children
of both sexes, 5-10 yr of age, born in agricultural areas
that perform biological pest control of the Sargent
Baez County, of the First District of the Cordillera
Department. Most of the adult population were
sugar cane producers, members of the Manduvirá
Cooperative (Cooperativa Manduvira)12. Families in
the San Juan Colony as well as those of the Sargent
Baez County lived in rural areas.
Exclusion criteria: Children without the consent of the
parents or guardians, all those children who refused to
take part in the study or those with physical disabilities,
292 INDIAN J MED RES, SEPTEMBER 2019
pubertal onset, chronic pathologies and congenital
malformations and with a history of X-ray exposure
in the past six months were excluded. Based on the
suggestions given by Preston and Hoffman13 the simple
size was calculated. For the present study, 43 cases and
41 controls were selected. The invitation to participate
in the study was extended through communication
with established community leaders. Subsequently,
only with those individuals who read, understood
and accepted the terms of the informed consent form
and additionally were not excluded by the exclusion
criteria, were studied. For the study of cell damage
and MNs, the unit of analysis was constituted by oral
mucosa cells and peripheral blood lymphocytes for the
comet assay.
Measurements and instrument used to compile data:
A questionnaire structured in six sections (risk factors,
sociodemographic characteristics, anthropometric
measurements, clinical profile, laboratory and
biomarker study) with closed questions, was developed
and validated. The quality control was done through
a ‘test-retest’ in a subsample of ten individuals, with
similar characteristics to the target population, that
were administered through interviews. In those
questions that presented difficulties of understanding,
a deeper investigation was carried out to facilitate the
analysis process and the corresponding adjustment of
the items, thus ensuring an optimum version that was
applied to the study sample.
To find about chronic exposure as a measure of the
effect, the MN test and the comet assay were utilized.
On the exposed population, the collection of genetic
material was done at a time when the fumigation with
pesticides was being carried out on a non-intensive
basis. Acute intoxication from organic phosphates
and carbamates was determined through plasma
cholinesterase on both populations of interest.
Sample collection
Urine and blood (5 ml) samples taken in
the morning (haemogram, proteinaemia, plasma
cholinesterase, renal, hepatic and thyroid profile) were
collected in January 2016 from group A children.
Material for the comet assay and MN test was collected
in August 2016. From group B children, urine and
blood samples were collected in September 2016, for
comet assay and MN test. The nutritional status was
determined by anthropometry: weight and height14;
proteinaemia, blood analysis, renal, hepatic, thyroid,
plasma cholinesterase, simple urine and serial faeces.
Anaemia was considered when Hb was below 11.5 g/
dl15 and hypoproteinaemia below 6 g/dl16.
Genotoxicity biomarkers: The bioassays employed
were carried out by the Laboratory of Genetic
Toxicology of the Faculty of Health Sciences at the
Catholic University Our Holy Mother of Asuncion.
Buccal micronucleus cytome (BMNcyt) assay: After
performing a mouthwash with water, the inner surface
of the cheek of the individuals was scraped with a sterile
wooden tongue blade, pre-moistened in physiological
serum. The sample was fixed on slide with 80 per cent
methanol for 1 h and then allowed to dry at room
temperature. The samples were stained according to
the Feulgen technique17, for which the hydrolysis was
done by immersing the sheets in 1N HCl at 60°C for
10 min. To cut off the hydrolysis, the samples were
washed in cold distilled water, and then placed in the
Schiff reagent (Sigma-Aldrich, USA) for 60 min.
For analysis, 2000 cells per individual were taken
according to the Tolbert technique18. The frequency
of MNs and other nuclear abnormalities such as
karyorrhexis (KR), broken egg (BE), karyolysis (KL),
pyknosis (PIk) and binucleated (BN) cells, were
determined based on the considerations of Bolognesi
et al6. The cells were evaluated with light-field
Olympus microscopes, model CX21, from Japan, with
a magnification of 1000×.
Comet assay: Evaluation of DNA damage was done by
the comet assay19,20. Forty microlitre of peripheral blood
was collected from the third finger of each participant
in Eppendorf tubes, containing 1.5 ml of cell culture
medium (RPMI, Roswell Park Memorial Institute), 20
mM EDTA (ethylenediaminetetraacetic acid) and 10
per cent dimethyl sulfoxide (DMSO), for correct assay.
Initial sample (120 μl) was mixed with 240 μl of 0.7
per cent low melting point (LMP) agarose. These were
plated on gelled sheets with one per cent normal melting
point agarose, and coated with a third layer of LMP
agarose. The gelled sheets were maintained at 4°C for 6
to 8 h immersed in a lysis solution (lysis buffer, Triton
X-100 and DMSO), for membrane lysis. The plates were
placed in an alkaline-buffered electrophoresis cuvette
at pH 12.5 for 20 min to allow the decomposition of
DNA and exposure of damaged alkali sites. After this,
electrophoresis was performed for 20 min at 25 V and
300 mA and, the sheets were washed in a 0.4 M Trizma
base at pH 7.5 neutralization buffer to remove excess
alkali and the detergents. Ethidium bromide (80 μl of
 LEITE et al: DNA DAMAGE IN CHILDREN 293

10 μg/ml) was used for staining; 20 min later, deionized Table. Demographic profile, nutritional status, eosinophilia
water was used to remove excess dye. The slides were and cholinesterase in the exposed and not exposed population
examined under epifluorescence microscope (ZEISS Variables Exposed Not
AXIOS A1, Germany) with an increase of 200×, using (n=43) exposed
a 590 nm filter. (n=41)
Age, mean±SD (yr) 7.6±1.8 8.0±1.8
Two hundred comets per participant were analyzed
using the Comet Imager software v2.2 (MetaSystems, Group, n (%)
Germany). Cell damage was determine using the 5‑6 13 (30.2) 10 (24.4)
following parameters into account: Tail length (TL): 7‑8 14 (32.6) 13 (31.7)
Total length of the comet, from the nuclear centre to 9‑10 16 (37.2) 18 (43.9)
the end of the tail. Tail moment (TM): Migration length Sex, n (%)
of the DNA outside the nucleus forming the tail of the Male 27 (62.7) 19 (46.3)
comet. Olive TM (OTM): Tail size of the comet plus
Female 16 (37.2) 22 (53.7)
the total DNA fraction from the centre of the head to
Nutritional status, n (%)
the tail end. Head DNA: the percentage of DNA in the
head of the comet. Tail DNA: the percentage of DNA Suitable stature 40 (93.0) 33 (80.5)
in the tail of the comet that indicates the amount of Risk low stature 3 (7.0) 8 (19.5)
fragmented DNA that migrated on electrophoresis. Low stature - -
Proteinaemia, g/dl (mean±SD) 7.2±0.5*** 7.5±1.1
Statistical analysis: For data analysis related to
demographic characteristics, nutritional status and Eosinophilia, % median (rank) 8.0±3.1*** 3.5 (2.5)
anaemia, frequency measures, central tendency and Hb, g/dl (mean±SD) 12.5±0.5 12.5±0.6
dispersion, were used depending on the nature of the Cholinesterase, U/l (mean±SD) 9182±1830 9493±1623
samples. Significance of the differences between groups ***
P<0.001 compared to not exposed group; Hb, haemoglobin
was analysed using the Student’s t test and Chi-square
test. Non-parametric Mann-Whitney test was used for
the evaluation of data related to DNA damage.

Results
The demographic characteristics are presented in
the Table. The children in the two groups had similar
age, haemoglobin level and nutritional status. They
all had normal hepatic, renal and thyroid function.
There was a significant difference in eosinophil count
between the two groups. No child presented with
chronic malnutrition (height/age); proteinaemia values
were whitin the normal range in both the studied
groups. One child (exposed group) was excluded from
the study because of chronic renal failure, detected at
the time of the study.
The results of the buccal MN cytome assay showed
highly significant differences when comparing the
exposed population with the unexposed population, as
shown in Fig. 1. The frequency of MN was higher in
the exposed group (6 vs 1) (P<0.001). Similar trends
are observed with BN cells (2.9 vs 0.5, P<0.001);
BE (5.5 vs 1.0, P<0.001), karyorrhexis (6.7 vs 0.5,
P<0.001); karyolysis (14.0 vs 1.0, P<0.001); pyknosis
Fig. 1. Anomaly rate in oral cells per 1000 cells of exposed (E)
(7.4 vs 1.2, P<0.001); and condensed chromatin (25.5 (n=43) and not exposed (NE) (n=41). P25, 25th percentile; P75,
vs 7.0, P<0.001). 75th percentile.
294 INDIAN J MED RES, SEPTEMBER 2019
Fig. 2. DNA damage, measured by length in μm of tail length, tail
moment and tail moment olive parameters of the comet assay of
exposed (n=43) and not exposed (n=41) populations.
The mean values of TL, TM and OTM on the cells
of the exposed children showed significant (P<0.001)
differences compared to those not exposed, as shown
in Fig. 2. The DNA percentage in the tails and heads of
the comets of exposed and unexposed children showed
a higher percentage of DNA in the head of the comets
of unexposed children compared to the exposed group
and higher percentage of DNA in the comet tails of
exposed children’s cells compared to the unexposed
(Fig. 3).
Discussion
The increase in TL, TM, OTM values and
the percentage of DNA in the tail of the observed
comets, in peripheral white blood cells of children
exposed to different concentrations of agrochemicals,
indicated that exposure to these compounds induced
DNA damage. An earlier study from Brazil showed
similar findings21. Studies in which the comet assay
was used have reported that organophosphates and
pyrethroids, commonly used as pesticides, have a
high genotoxic potential22. Others have indicated
glyphosate and organophosphates as pesticides that
induce DNA damage23. Glyphosate has been reported
to be safe for humans24. The continued exposure and
persistence of unrepaired damage induced by pesticide
and agrochemical components and the formation
of free radicals may induce increased chromosomal
aberrations, and the complex mixture of chemical
components would interfere with DNA repair
mechanisms25.
The buccal MN cytome test revealed damage in the
genetic and cytotoxic material in the cells of exposed
children, manifested by the increase of MN, BE and BN
cells, compared to the population of unexposed children.
The MN rate per 1000 cells counted in unexposed
children was 0.22 per cent; according to Bonassi et al26,
Fig. 3. DNA distribution according to the comet assay for exposed
(n=43) and not exposed (n=41) populations.
the baseline MN frequency for individuals not exposed
to genotoxic agents is from 0.3 to 1.70. Though our
study was conducted on children, our data for unexposed
children were within the rank found by Bonassi et al26.
Other nuclear figures have been observed in a large
number in cells of exposed children, such as condensed
chromatin, associated with apoptosis processes and
corresponding to regions with little transcriptional
activity6,27, karyolysis, which corresponds to the state
of total disintegration of the nucleus and occurs in
late states of apoptosis and necrosis28. These findings
were similar to a previous investigation in which
significant differences were found in the frequencies
of MN, BN cells and karyorrhexis in a population of
children exposed for six years to contaminants from
an agrochemical factory, compared to the non-exposed
population29.
The exposed population was surrounded by large
soybean crops, at a distance of 20 km of the nearest
urban centre with which it was connected through a
dirt road, and air pollution due to vehicular use was
scarce. This population was characterized by an overall
good health and nutritional state. These children were
exposed to intensive spraying and pesticide use in their
homes. The level of damage at the DNA level, utilized
as a marker of the effect in this study, was significant
when comparing the exposed and non-exposed groups.
There were no significant differences in relation
to the demographic profile of exposed group children
when compared with the children of the unexposed
population. The main confounders such as anaemia and
malnutrition were not found. An increase in eosinophil
count was identified in the exposed population that
was not associated with infestations by helminthic
parasites30. Verea et al31 described a significant increase
 LEITE et al: DNA DAMAGE IN CHILDREN 295
in eosinophils and a decrease in white blood cells in a
study carried out with a group of agricultural workers
exposed to pesticides.
In children younger than two years, the
mechanisms of hepatic detoxification mediated by
glutathione S-transferase and glucuronyltransferase as
well as by the cytochrome P450 enzyme system are
immature, and their ability to inactivate and detoxify
will determine whether a chemical compound will be
toxic or not. Glyphosate can cause interference with
these enzymes and alter their homeostasis.
The results found in several investigations of
Latin America and Europe5,32 constitute evidence
that indicate that the exposure to pesticides causes
genotoxic damage, and this allows to establish
the scientific bases to realize a preventive health
intervention, especially in more vulnerable populations
such as pregnant women and children. This genotoxic
damage can initiate the process of carcinogenesis,
mutagenesis and teratogenesis. Several pesticides
have been characterized as probable human
carcinogens by the International Agency for Research
on Cancer33 associated with exposure to pesticides in
epidemiological studies4.
In conclusion, a greater genotoxic and cytotoxic
effect was observed in children exposed to pesticides
compared to non-exposed children. There was a
greater DNA damage in children exposed to pesticides
compared to those not exposed. Further studies need to
be done on exposure to the mixtures of pesticides and
their effects on health over time.
Financial support & sponsorship: This investigation
was conducted with the financial support of the National Council
on Science and Technology (CONACYT) Project: 14INV180,
Paraguay.
Conflicts of Interest: None.
References
1. The Community Action Program of East Central Oregon.
Ranking mundial. Available from: http://capeco.org.py/
ranking-mundial-es/, accessed on April 13, 2017.
2. Franceschelli I. [Importación de agroquímicos: La principal
actividad económica nacional no es nacional]. In: Marielle P,
editor. [Con la soja al cuello 2016: Informe sobre agronegocios
en Paraguay]. Asunción, Paraguay: BASE-Is; 2016. p. 28-31.
3. Benítez-Leite S, Corvalán R, Avalos DS, Almada M,
Corvalán A. Violated rights in rural populations exposed to
transgenic soybean crop (Preliminary Study). Br J Med Med
Res 2016; 16 : 1-8.
4. Bolognesi C, Holland N. The use of the lymphocyte
cytokinesis-block micronucleus assay for monitoring
pesticide-exposed populations. Mutat Res 2016; 770 : 183-203.
5. Aiassa D, Mañas F, Bosch B, Gentile N, Bernardi N, Gorla N.
[Biomarcadores de daño genético en poblaciones humanas
expuestas a plaguicidas]. Acta Biol Colomb 2012; 17 : 485-510.
6. Bolognesi C, Knasmueller S, Nersesyan A, Thomas P,
Fenech M. The HUMNxl scoring criteria for different cell
types and nuclear anomalies in the buccal micronucleus
cytome assay – An update and expanded photogallery. Mutat
Res 2013; 753 : 100-13.
7. Bolognesi C, Knasmueller S, Nersesyan A, Roggieri P,
Ceppi M, Bruzzone M, et al. Inter-laboratory consistency and
variability in the buccal micronucleus cytome assay depends
on biomarker scored and laboratory experience: Results from
the HUMNxl international inter-laboratory scoring exercise.
Mutagenesis 2017; 32 : 257-66.
8. Collins A, Koppen G, Valdiglesias V, Dusinska M,
Kruszewski M, Møller P, et al. The comet assay as a tool for
human biomonitoring studies: The comNet project. Mutat Res
Rev Mutat Res 2014; 759 : 27-39.
9. Neri M, Bonassi S, Knudsen LE, Sram RJ, Holland N,
Ugolini D, et al. Children’s exposure to environmental
pollutants and biomarkers of genetic damage. I. Overview and
critical issues. Mutat Res 2006; 612 : 1-3.
10. Valiente H. [Comunidades en lucha: Cuatro demandas al
Estado paraguayo por violación de Derechos Humanos].
Asunción, Paraguay: BASE-Is; 2014. p. 128.
11. United Nations. Report of the Special Rapporteur on the right
to food on her mission to Paraguay A/HRC/34/48/Add.2.
Geneva: General Assembly; 2017.
12. Cooperativa Manduvirá. [Primera Cooperativa productora y
exportadora de azúcar orgánica del Paraguay]. Asunción:
Cooperativa Manduvira Ltda; 2012.
13. Preston RJ, Hoffmann GR. Genetic toxicology. In: Klaassen CD,
editor. Cassaret & Doull’s toxicology: The basic science of
poisons, 7th ed. Nueva York: McGraw Hill; 2008. p. 381-413.
14. World Health Organization. WHO anthro survey analyser and
other tools. Available from: https://www.who.int/childgrowth/
software/es/, accessed on April 30, 2017.
15. World Health Organization. Haemoglobin concentrations
for the diagnosis of anaemia and assessment of severity
(WHO/NMH/NHD/MNM/11.1). Geneva: WHO; 2011.
16. Manary MJ, Trehan I. Protein-energy malnutrition. In:
Goldman L, Shafer AI, editors. Goldman-Cecil Medicine. 25th
ed. Philadelphia, PA: Elsevier Saunders; 2016.
17. Jalayer Naderi N. Reporting an experience: Improving the
Feulgen staining technique for Better visualizing of nucleus.
Iran J Pathol 2018; 13 : 106-7.
18. Tolbert PE, Shy CM, Allen JW. Micronuclei and other nuclear
anomalies in buccal smears: Methods development. Mutat Res
1992; 271 : 69-77.
19. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple
technique for quantitation of low levels of DNA damage in
individual cells. Exp Cell Res 1988; 175 : 184-91.
20. Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S,
Burlinson B, Clay P, et al. Recommendations for conducting
296 INDIAN J MED RES, SEPTEMBER 2019
the in vivo alkaline comet assay 4th International Comet Assay
Workshop. Mutagenesis 2003; 18 : 45-51.
21. Benedetti D, Nunes E, Sarmento M, Porto C, Dos Santos CE,
Dias JF, et al. Genetic damage in soybean workers exposed to
pesticides: Evaluation with the comet and buccal micronucleus
cytome assays. Mutat Res 2013; 752 : 28-33.
22. Kaur R, Kaur S, Lata M. Evaluation of DNA damage in
agricultural workers exposed to pesticides using single
cell gel electrophoresis (comet) assay. Indian J Hum Genet
2011; 17 : 179-87.
23. Kwiatkowska M, Reszka E, Woźniak K, Jabłońska E,
Michałowicz J, Bukowska B. DNA damage and methylation
induced by glyphosate in human peripheral blood mononuclear
cells (in vitro study). Food Chem Toxicol 2017; 105 : 93-8.
24. Williams GM, Kroes R, Munro IC. Safety evaluation and
risk assessment of the herbicide roundup and its active
ingredient, glyphosate, for humans. Regul Toxicol Pharmacol
2000; 31 : 117-65.
25. Bolognesi C. Genotoxicity of pesticides: A review of human
biomonitoring studies. Mutat Res 2003; 543 : 251-72.
26. Bonassi S, Coskun E, Ceppi M, Lando C, Bolognesi C,
Burgaz S, et al. The Human micronucleus project on exfoliated
buccal cells (HUMN(XL)): The role of life-style, host factors,
occupational exposures, health status, and assay protocol.
Mutat Res 2011; 728 : 88-97.
27.
Oberhammer FA, Hochegger K, Fröschl G,
Tiefenbacher R, Pavelka M. Chromatin condensation during
apoptosis is accompanied by degradation of lamin A+B,
For correspondence: Dr Stela Benitez Leite, Alférez Juan Manuel Iturbe 451 c/Acuña de Figueroa, Asuncion, Paraguay
e-mail: benitezleitestela@gmail.com
without enhanced activation of cdc2 kinase. J Cell Biol
1994; 126 : 827-37.
28. Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S,
Zeiger E, Knasmueller S, et al. The micronucleus assay
in human buccal cells as a tool for biomonitoring DNA
damage: The HUMN project perspective on current status and
knowledge gaps. Mutat Res 2008; 659 : 93-108.
29. Benítez-Leite S, Macchi ML, Fernández V, Franco D,
Ferro EA, Mojoli A, et al. Cellular damage in a child
population potentially exposed to pesticides [Daño celuar en
una población infantil potencialmente expuesta a pesticidas].
Pediatr (Asunción) 2010; 37 : 97-106.
30. Desvars P, Avalos DS, Galeano A, Benítez-Leite S,
Campuzano A. Prevalence of incidental finding eosinophilia
in children in rural communities [Prevalencia de eosinofilia
de hallazgo incidental en niños de comunidad rural]. Pediatr
(Asunción) 2016; 43 (Suppl) : 62.
31. Verea MC, Masoero C, Gentile N, Bosch B, Aiassa D. Possible
biomarkers to assess occupational exposure to pesticides
[Biomarcadores posibles para evaluar la exposición laboral
a plaguicidas]. Retel Rev Toxicol Línea 2016; 45 : 13-27.
32. Koller VJ, Fürhacker M, Nersesyan A, Mišík M, Eisenbauer M,
Knasmueller S, et al. Cytotoxic and DNA-damaging properties
of glyphosate and roundup in human-derived buccal epithelial
cells. Arch Toxicol 2012; 86 : 805-13.
33. International Agency for Research on Cancer. Some
organophosphate insecticides and herbicides: IARC
monograph on the evaluation of carcinogenic risk to
humans;vol 12. Geneva: World Health Organization; 2017.